The Hope laboratory has recently published the identification and characterization of the earliest cells infected by SIV in the mucosa 48 hours after vaginal and rectal inoculation. In developing this system, the Hope laboratory has developed critical methods and expertise allowing the study of SIV and immune target cell populations within the mucosal barriers. These capabilities will be leveraged along with a strategy to specifically seed infection in tissue so that the latent reservoir will be generated within a discrete area at these mucosal sites (and other body sites). The specific seeding of the reservoir can be validated and complemented by the unique PET/CT imaging expertise/infrastructure pioneered by Francois Villinger to detect foci of active virus infection. With the knowledge of mucosal reservoir localization, we will also track SIV rebound from latency in the early reservoir cells as it cascades to viremia when early, suppressive ART is withdrawn. Identification of these early events will be aided by bioluminescent reporter viruses and whole animal PET/CT. These approaches will provide critical new insights into latently infected cells and facilitate the development of a cure to HIV infection. The proposed studies will achieve the following specific aims: (1) Define and characterize the mucosal virus reservoir cells (cells harboring SIV that remain persistent/latent during suppressive ART). This will specifically characterize the cells with persistent virus that are established during the earliest aspects of acute SIV infection of macaques. This aim will determine the phenotypes, locations, viral production status, and persistence within the mucosa of these reservoirs established with the first days of infection before starting suppressive ART. (2) Define and characterize the spread of virus within and beyond the mucosal foci of SIV infected cells, and from which rebound viremia originates, after stopping early, suppressive ART. This aim uses using both PET/CT and reporter viruses, and will also evaluate host responses and biomarkers. (3) Determine the stability of the mucosal reservoir over time and evaluate the effects of catalytic mTOR inhibitors (given before and temporarily after stopping ART) on the SIV viremia rebound from mucosa, following 24 weeks of viral suppression by ART that started in the first days after mucosal inoculation of SIV. We will utilize recently developed methods and expertise to observe the formation of the mucosal reservoir formed in the first few days after virus acquisition. By seeding the reservoir in specific areas we should be able to observe the rebound from latency. A better understanding of the localization and cell type of the reservoir cells will facilitate the development of a cure for HIV.